A developed thin-layer chromatography method for the quantification of stigmasterol in leaves, stem and root of Sida cordifolia Linn., assessment of secondary metabolites and in vitro antioxidant activity
摘要
Stigmasterol, a phytosterol belonging to the class of tetracyclic triterpenes is present in Sida cordifolia. The present study was undertaken to develop a simple, rapid, accurate, and validated thin-layer chromatography (TLC) method for the simultaneous detection and quantification of stigmasterol in the leaves, stem and root of S. cordifolia. The chromatographic separation of standard stigmasterol and plant samples was achieved on TLC aluminum plates precoated with silica gel 60F254 using the mobile phase n-hexane‒ethyl acetate (8:2, V/V). To visualize the band, the developed plate was dried with hot air to remove the mobile phase, then derivatized with freshly prepared anisaldehyde‒sulfuric acid reagent and heated at 105 °C for 5 min on a CAMAG TLC plate heater. Densitometric scanning was performed at 540 nm. The regression equation showed a linear relationship, with a value of r2 = 0.986. The retardation factor (RF) value for stigmasterol was achieved as 0.35 ± 0.001. The method was validated following the International Council for Harmonisation (ICH) guidelines showing remarkable performance. Findings confirmed that the maximum content of stigmasterol was found to be in the sample of Amarkantak range leaf (1.109 ± 0.01%) > root (0.712 ± 0.076%) > stem (0.544 ± 0.038%) as compared with other locations. In phytochemical examination, maximum total phenol content (TPC), total flavonoid content (TFC), total tannin content (TTC), and total alkaloid content (TAC) content were observed in the samples of Amarkantak range [33.46 ± 1.89 mg of gallic acid equivalent per gram dry weight (mg GAE/g DW)] in leaves, [98.86 ± 0.51 mg quercetin equivalents per gram dry weight (mg QE/g DW)] in stem, [32.41 ± 1.16 mg of tannic acid equivalent per gram dry weight (mg TAE/g)] in leaves and [10.63 ± 0.63 mg of colchicine equivalent per gram dry weight (mg CE/g DW)] in root, respectively. Physicochemical properties of different parts (root, stem, and leaves) were assessed demonstrating statistically significant variations (p < 0.05) across all parameters. The root recorded the highest total ash content (2.21 ± 0.08 mg/g), whereas the leaves exhibited the maximum acid-insoluble ash (0.63 ± 0.30 mg/g). Water-insoluble ash was significantly higher in the stem (2.36 ± 0.20 mg/g), while the highest sulphated ash was found in the leaves (2.76 ± 0.05 mg/g). Extractive values also varied notably, with the stem showing the highest water-soluble (6.46 ± 0.25 mg/g) and alcohol-soluble (4.76 ± 0.05 mg/g) extractives. Leaves demonstrated higher antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) assays with IC50 values of 37.19 ± 9.83 μg/mL, 45.49 ± 15.7 μg/mL and dose-dependent increase in absorbance 0.563 ± 0.001 in reducing power assay (RPA) as compared with root and stem. The present study depicts the utility of the developed method in herbal preparations containing stigmasterol.